Introduction: Despite an information explosion about antimicrobial resistance mechanisms in bacteria, patients are dying of untreatable infections for the first time since antibiotics were discovered. The main cause of multidrug resistance (MDR) in gram-negative bacteria is active efflux of drug by bacterial pumps. Knowing how the pumps are regulated is the key to understanding MDR development: bacteria can become MDR simply by increasing expression of these efflux pumps. Significance: Bacteroides fragilis are ubiquitous gut commensals that cause devastating disease when they escape the gut; their resistance to antimicrobials is rising significantly. In aerobes, MDR is caused by efflux pumps regulated by transcription factors (TFs). We hypothesize that MDR efflux pumps in B. fragilis are similarly regulated by TFs. These TFs, and the pumps they regulate, may also regulate other virulence factors. VA patients are particularly at risk for anaerobic infections. Mortality in Bacteroides bacteremia is high (25- 50%) and liver disease, common in VA patients, is a risk factor for increased mortality. We identified several MDR clinical isolates (including one from a GI in Afghanistan) with increased pump activity; these isolates were resistant to metronidazole and other agents and resulted in one death and one amputation, respectively. Preliminary Studies: In the previous funding period, we identified and characterized the B. fragilis bme efflux pump genes and showed that they were implicated in clinical MDR (described in 23 publications); we found that increases in pump levels and in resistance could be induced by a variety of agents and stressors including antimicrobials, salicylate, bile and quorum sensing molecules. We constructed new vectors and modified them to mimic the molecular tools available for use with aerobes. Research Plan: Our long term goal is to unravel the complex regulation of MDR in B. fragilis. Specifically, our goals now are to: AIM 1: Identify the TFs most important in efflux-mediated MDR (both activator and repressor TFs) using a functional genomic approach. AIM 2: Characterize TFs most important in efflux-mediated MDR. Determine the phenotype associated with the TF, measure MICs (minimal inhibitory concentrations) and determine the relevant bme genes. Demonstrate binding by physical and functional approaches. AIM 3: Determine the clinical prevalence of TF regulation in efflux mediated MDR. Measure TF transcription levels in clinical isolates, including the recently acquired MDR strains. Determine the effect of TF on efflux-mediated MDR and on ability to form biofilms. Assess the effect of bile exposure on TF transcription levels, MICs and biofilm forming ability. This study takes advantage of our unique resources: expertise with molecular and biochemical approaches, access to a massive collection of clinical isolates and associated MIC data, and excellent collaborative relationships with world-class experts. Once completed, this work, together with the studies already conducted, will reveal important new information about regulation of BF MDR pumps and will greatly contribute to our knowledge of regulation mechanisms in this bacterium that is subject to very different stresses than aerobic pathogens. B. fragilis is an important component of the gut microbiome (which has been called a separate organ within the human body) that is recognized as having its own impressive metabolic profile and profoundly affects almost every aspect of human health and disease. In this global environment, there is little time from the emergence of the first MDR strains to widespread resistance. Racing to develop effective drugs when untreatable infections emerge is not a viable public health strategy. Regulators of MDR efflux are important targets in drug discovery; identifying and understanding the TFs that are most important in these MDR organisms will lead to the development of new and effective interventional therapies that target the expression or translation of these factors.